Cells low in ROCKI (center) lack stress fibers.

The mammalian rho kinases, ROCKI and ROCKII, control distinct cytoskeletal functions from distinct locations, report Yoneda et al. on page 443. ROCKI is required for formation of actin stress fibers and cell adhesion, whereas ROCKII is needed for phagocytosis.The ROCK proteins are major effectors of the Rho signaling pathway, controlling cell adhesion, motility, and other actin-based functions. In kinase overexpression assays, ROCKI and ROCKII appear redundant, but several experiments, including mouse knock-outs, hinted that the proteins have unique duties despite their structural similarity.

While studying a signaling pathway that links integrins and cell surface proteoglycans to adhesion, Yoneda et al. saw that ROCKI but not ROCKII activity increased during adhesion. To learn how the proteins differ functionally, the team knocked down each ROCK gene independently using siRNA sequences that left the other gene unchanged. Cells lacking ROCKI formed few stress fibers or focal adhesions. ROCKII-depleted cells produced oversized stress fibers and adhesions, and showed problems with phagocytic uptake of fibronectin-coated beads.

The PH domain is the most divergent region of the two ROCKs, and GFP-PH from each protein localized to distinct subcellular locations. The ROCKI PH domain was evenly distributed at membranes, whereas ROCKII PH concentrated in ruffles at the cell surface. Subsequent analysis showed that only ROCKII PH binds to PIP3 lipids and may, therefore, be regulated by PI3-kinase.

Although the ROCK proteins phosphorylate similar targets in vitro, Yoneda et al. think that subcellular targeting restricts the pair's functions. This is consistent with overexpression of either protein masking their differences. If the team is right, then what holds for real estate holds for the ROCKs: it's all about location, location, location.